Advanced Live-Cell Imaging station for multipurpose biomedical imaging

Aim: To establish an integrated, computer-driven, confocal laser scanning microscope (CLSM)-based imaging station to perform optimal 4D (space + time) imaging of a wide array of biomedical events in (single) living cells. The proposed system integrates standard one-pinhole CLSM with novel spinning-disc technology and a monochromator system for ultra-rapid multicolor imaging. The CLSM is equipped with a recently introduced 32PMT detector for spectral analysis that allows for the simultaneous recording of multiple colors of green-fluorescent protein (GFP)-based probes without 'bleedthrough', a pre-requisite for co-localization studies and inter-molecular fluorescence resonance energy transfer (FRET). The spinning disc scan-head importantly increases the possibilities of live-cell imaging, because it minimizes bleaching and photo toxicity thus allowing to study cells over prolonged time periods. These complementary systems combine latest technology and together cover a wide and novel spectrum of biomedical applications, ranging from an accurate visualization of fast intracellular dynamics to the continuous registration of slow biological processes over long periods (over 24h).Innovative aspects: Live cell imaging is a powerful new technology that literally reveals the kinetics and direction of cellular dynamics, which can not be deduced from static images. This technical progress will certainly pave the way for breakthroughs in our understanding of the cell's organization and regulation. The integration of multiple state-of-the-art imaging techniques on a single station allows for the exploitation of a broad spectrum of biomedical applications, which cannot be performed on currently available apparatuses. The proposed station will be positioned in the Utrecht Medical Cell Imaging Center (UMCIC) of the UMC-U Cell Biology Department, where recently a technique for correlative live cell - immuno-electron microscopy (IEM) was developed. Herewith, a fluorescent protein identified in live cells is subsequently visualized by high-resolution IEM. This approach provides important surplus value, because it integrates kinetics and localization information of a single protein or organelle against an ultrastructural background. The proposed combination of technology will be unique in the world and complement the array of visualization techniques for cell biological purposes at the Utrecht University campus.Scientific significance: To date an increasing number of diseases is recognized as an intracellular transport defect. Examples include diabetes, obesity, cancer, neurodegenerative diseases (e.g. Alzheimer's disease) and infectious diseases. The Klumperman and Bos groups focus their research on two important and inseperable facets of intracellular transport: vesicular trafficking and cytoplasmic signalling. Clearly, to study dynamic intracellular transport events imaging of live cells is mandatory. The proposed equipment will be used to capitalize on the know-how and expertises of the two groups and to bridge the two research fields. More specifically, live cell imaging - in combination with correlative IEM - will be used to study 1. Molecular mechanisms and transport kinetics of machinery and Alzheimer-related proteins through the cell. 2. Monitor the formation of protein complexes by Ras-like small GTPases in time and space. In addition, the station will provide the necessary means to study a wide variety of biomedical applications by research groups at the UMC-U, the Academic Biomedical Cluster, as well as at the Utrecht graduate Schools 'Institute of Biomembranes' and 'Developmental Biology'. The UMCIC will enable these groups to exploit the unique combination of live cell imaging and IEM.